Rahman M. Mizanur

Convergent Synthesis of Homogeneous Glc1Man9GlcNAc2-Protein and Derivatives as Ligands of Molecular Chaperones in Protein Quality Control

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.

Bacterial CMP-sialic acid synthetases: production, properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell–cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5′-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.

Recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from Pyrococcus furiosus.

Alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-D-glucose-1-phosphate. We report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 degrees C and pH 6.5-7. This archaeal protein retained its complete activity after 24 h at 80 degrees C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 degrees C in the presence of 10 mM maltoheptaose and at 10 mg ml(-1) glycogen concentration was 52 U mg(-1) and 31 U mg(-1), respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 +/- 2 mM and 0.60 +/- 0.02 mM min(-1) microg(-1), respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.

Glycosidase activity profiling for bacterial identification by a chemical proteomics approach

Genome sequencing projects are suggesting there are dozens of glycosidase sequences that could be used to fingerprint cell types and serve as starting points for biocatalyst discovery. Herein, we present a simple chemical proteomics approach to profile intracellular glycosidase activities of three different bacterial cell extracts using a synthetic α- and β-linked library of 18 representative substrates with electrospray ionization-mass spectrometry (ESI-MS) reaction monitoring. Three target bacteria – Escherichia coli K12, Bacillus cereus and Pseudomonas aeruginosa – can be easily differentiated by this method. Compared with traditional chromogenic and fluorogenic methods to profile bacterial enzyme activities individually, this MS-based method can detect multiple enzyme activities in one reaction and easily highlight activity differences between whole cell extracts.